Fig. 3

PITX2C promotes cell mobility and self-renew of HCC. A Representative images (left) and bar chart (right) of cell migration and invasion abilities in LO2, MiHA and Hep3B cells with PITX2A, 2B, 2C-transfection by Transwell (50,000 cells) and Matrigel invasion (100,000 cells) assays. Migrated and invaded cells were stained with crystal violet and counted under a microscope. Values indicate the mean ± SD of three independent experiments (*P < 0.05; **P < 0.01; independent Student t test). B Western blotting analysis was used to compare expressions of epithelial and mesenchymal markers between PITX2A, 2B, 2C-transfected cells and control cells. β-actin was used as loading control. C Spheroid formation assay was used to evaluate the self-renewal ability of PITX2A, 2B, 2C-transfected and control cells (left). The numbers of primary and secondary spheroids are calculated in the bar chart (right). Values indicate the mean ± SD of three independent experiments (*P < 0.05, **P < 0.01, independent Student’s t-test). D Representative images of organoids derived from two HCC patients. The mRNA levels of PITX2C and AFP were validated by qRT-PCR. PDO. Patient derived organoid; PT. primary tumor tissue. PT is used as control. Data are presented as the mean ± SD of 3 independent experiments (*P < 0.05, **P < 0.01, independent Student’s t-test). E Relative expression of AFP and Lgr5 in PITX2C and shPITX2- transfected cells (Right: non-transfected cells were used as controls). Data are presented as the mean ± SD of 3 independent experiments